Sphingomonas strains, such as ATCC 53159 and ATCC 31461, produce copious amounts of capsular polysaccharide. While under some conditions polysaccharide may be released from the cell [5, 6], during growth with abundant carbon source as in fermentation, the polysaccharide is firmly attached to the cell surface. Attempts to increase productivity of fermentations for diutan and gellan may be limited by the capsular nature of the polysaccharide, which may impair uptake of nutrients. Also, if there are a limited number of sites for biosynthesis of the polysaccharide, there may be a maximum amount of polysaccharide that can be produced by each cell. The polysaccharide gellan has been observed to be involved in cell clumping since mutants that do not make any polysaccharide grow uniformly in suspension [3]. These cell clumps may interfere with techniques such as determination of cell number by optical density, centrifugation of cells, e.g., for isolation of DNA or protein, and separation or lysis of cells for polysaccharide purification.
The mechanism of attachment and the genes involved in attachment of polysaccharide to the cell surface in Sphingomonads have not been previously determined. Induced mutants of Sphingomonas strains ATCC 31461, ATCC 31555, ATCC 31554, and ATCC 21423 that produce polysaccharide in a slime form have been isolated, but the genes mutated were not determined, and the methods of inducing and selecting the mutants were not disclosed [10]. Genes for biosynthesis of gellan [3, 8], diutan [1] and sphingan S-88 [9] have been isolated. The functions of many of these genes were assigned by biochemical tests or by homology to genes of known functions in databases such as GenBank. For example, genes have been identified that are involved in assembly of the tetrasaccharide repeat unit [7, 8], and in synthesis of the precursor dTDP-L-rhamnose [3, 9]. It would be expected that genes affecting only attachment of polysaccharide to the cell surface would still have the polysaccharide producing phenotype (i.e., mucoid colonies on solid media and viscous broth).
A cluster of 18 genes for gellan biosynthesis spanning 21 kb was described, in addition to four genes for gellan synthesis not in the cluster Harding N. E., Y. N. Patel, and R. Coleman, 2004. Organization of genes required for gellan polysaccharide biosynthesis in Sphingomonas elodea ATCC 31461. J Ind Microbiol Biotech 31:70-82, reference 3. The DNA sequences were deposited in GenBank in June 2003 (Accession number AY217008). Among the genes in the cluster were gelM, gelN, and gelI. A deletion of most of adjacent genes gelM and gelN was constructed. The gelI gene was inactivated by an insertion. The gelM-gelN deletion strain and the gelI mutant were shown to produce somewhat reduced amounts of gellan and more fluid broths, and the gellan produced was shown to have the same composition as gellan from the wild-type strain. The attachment of the polysaccharide to the cell was not reported
The Sphingomonas elodea gelR, gelS, and gelG genes appear to be in an operon in the same order as in the S-88 sps gene cluster, but not adjacent to the genes in the cluster of 18 genes (reference 3). The GelR protein was somewhat smaller than its S-88 homolog (659 vs. 670 amino acids) with 49% identity, and had homology to surface layer proteins and other membrane proteins. The DNA sequences of gelR, gelS and gelG genes were deposited in GenBank in June 2003 (Accession number AY220099). No mutation in gelR was constructed in this report (reference 3). Yamazaki et al. report that strains with mutations in gene spsR were still mucoid, indicating that they produce polysaccharide, but the polysaccharide was not characterized as to rheology or attachment to the cell ([9] and T. J. Pollock et al. DNA segments and methods for increasing polysaccharide production. U.S. Pat. No. 5,854,034.
Yamazaki described classical mutants of four Sphingomonas strains that produce polysaccharide as slime rather than attached to the cell. U.S. Pat. No. 6,605,461. Yamazaki did not describe how to screen mutagenized cultures for the slime phenotype. Yamazaki did not identify which gene or genes were mutated.
Sa-Correia reviewed work done on isolation of genes for gellan synthesis. Sa-Correia I., A. M. Fialho, P. Videira, L. M. Moreira, A. R. Marques and H. Albano. 2002. Gellan gum biosynthesis in Sphingomonas paucimobilis ATCC 31461: Genes, enzymes and exopolysaccharide production engineering. J Ind Microbiol Biotechnol. 29: 170-176. Sa-Correia described partial sequencing of some genes including urf32 and urf26 (=gelM and gelN of reference 3 above). The complete sequences of these genes were deposited in GenBank in April 2003 (GenBank Accession number AY242074). No function of these genes is reported. In the GenBank submission, genes urf32 and urf26 were merely designated as putative membrane protein and putative exported protein, respectively. No sequence for gelI or gelR was deposited.
Coleman describes the isolation of genes for diutan biosynthesis and investigation of some gene functions. R. Coleman, 2001, Cloning and analysis of Sphingomonas sp. ATCC 53159 polysaccharide genes. Master's Thesis, San Diego State University. The dpsM and dpsN genes, which were designated by Coleman as orf3 and orf4, were described but functions were not indicated.
A cluster of genes for biosynthesis of the S-88 polysaccharide from Sphingomonas strain ATCC 31554 was described. Yamazaki M., L. Thorne, M. Mikolajczak, R. W. Armentrout and T. J. Pollock. 1996. Linkage of genes essential for synthesis of a polysaccharide capsule in Sphingomonas strain S88. J Bacteriol 178: 2676-2687 and U.S. Pat. No. 5,854,034. The functions of genes urf32 and urf26 (homologs of dpsM, gelM and dpsN, gelN), and spsI (homolog of gelI, dpsI) were not described. Gene spsR (homolog of gelR, dpsR) was described as encoding a protein remotely similar to bacterial and fungal polysaccharide lyases The DNA sequences were deposited in GenBank (Accession number U51197).
There is a continuing need in the art to improve methods of making industrially useful sphingans and the properties of the sphingans.